The term “mesenchymal stem cells” refers to cells that are involved in the development and growth of the skeletal system in their embryonic stage and have the capability to differentiate into various kinds of skeletal cells, including osteocytes, chondrocytes, myocytes, fibrocytes, etc., after completion of growth. The mesenchymal stem cells are also referred to as bone marrow stromal stem cells. Unlike embryonic stem cells, mesenchymal stem cells do not cause tumors or ethical issues, and thus are highly useful for clinical applications.
However, the problems to be solved in the transplantation of stem cells are developing technology for facilitating engraftment (improving adaptability) and proliferation in vivo after transplantation and ensuring the capability to differentiate selectively into a desired tissue. To improve adaptability in vivo after transplantation, immune-related drugs are administered after transplantation. However, these drugs are administered to improve the adaptability of a subject to which stem cells are transplanted. A substance for improving the adaptability of the cells transplanted has not been reported yet.
The stem cells transplanted by the surrounding tissue environment undergo senescence, die out, and thus cannot exhibit effects of transplantation. In addition, smooth proliferation of the transplanted stem cells may be the first consideration for regeneration of the cells. In addition to engraftment and proliferation, a substance for stimulating selective differentiation into a desired tissue (bone tissue in the present invention) has also not been reported. Transcription factors essential in the differentiation of mesenchymal stem cells into osteoblasts and adipocytes are Runx2 and peroxisome proliferator-activated receptor-gamma (PPAR-gamma). These factors induce mesenchymal stem cells to differentiate into osteoblasts which produce bones and adipocytes. Runx2 is a protein which is observed to be localized to bone when injected externally, and in addition to this protein, several proteins are known to stimulate the differentiation of cells into bone tissue. Particularly, Hong et al. demonstrated that a 14-3-3-binding protein, TAZ (transcriptional coactivator with PDZ-binding motif), represses PPAR gamma transcription and activates Runx2 (Hong J. H. et al., Science, 309:1074, 2005). These proteins act to activate genes for osteogenic differentiation in the cytoplasm. However, the molecular weight of these proteins is too large to be introduced into the cytoplasm or tissue for a specific purpose (substances having a molecular weight of 600 or more are almost impossible to pass through the cell membrane). For this reason, the use of carriers to introduce these proteins into cells is acutely required.
Recently, the development of osteogenesis stimulators for repairing bone damaged by osteoporosis, bone fracture or surgery has been required. However, osteogenesis stimulators, such as bisphosphonates, calcitonin, estradiol or vitamin D, developed to date, mainly aim to inhibit bone resorption, and are not significantly effective to the regeneration of lost bone. Thus, many efforts have been made to discover new drugs that can stimulate osteogenesis.
In recent years, it was found that Tat (transactivator of transcription) protein, a kind of human immunodeficiency virus type-1, efficiently passes through the cell membrane and migrates into the cytoplasm. This function appears because of the characteristics of the protein transduction domain (PTD) of the Tat protein, and the exact mechanism thereof is not known yet (Frankel, A. D. and Pabo, C. O., Cell, 55:1189, 1988; Green, M. and Loewenstein, P. M., Cell, 55:1179, 1988; Ma, M. and Nath, A., J. Virol., 71:2495, 1997; Vives, E. et al., B. J. Biol. Chem., 272:16010, 1997).
Meanwhile, since it was found that a fusion protein obtained by linking PTD to other peptides or proteins is efficiently transported into cells, various applications using PTD has been attempted (Korean Patent Registration No. 10-0568457). However, there has not yet been an attempt to apply the transcription factor NF-Ya or antioxidant protein (e.g., SOD, thioredoxin, etc.) linked to a cell-penetrating peptide to tissue engineering for the purposes of promoting the engraftment of mesenchymal stem cells and inducing the proliferation thereof.
Accordingly, the present inventors have made extensive efforts to develop a cell permeable fusion protein for strengthening regenerative potential of stem cells, and as a result, have prepared a fusion protein by linking a cell-penetrating protein to the amino terminal of an antioxidant protein or a protein having the ability to improve cell proliferation, and have found that the fusion protein can increase the success rate of transplantation of stem cells, can be used to prevent and treat bone disease by selectively inducing the regeneration of bone tissue, can efficiently deliver a protein that inhibits adipocytes into cells, is easily synthesized, and causes no toxicity problem, thereby completing the present invention.